Turnover of Proteins as a Controller of Soil Nitrogen Cycling

自然生态系统和管理生态系统中的植物生产力通常受到氮（N）的限制，植物从土壤中获得的氮主要是铵或硝酸盐-两者都是氮的无机形式。然而，土壤中95%以上的氮以有机形式存在，主要是来自腐烂植物材料或分解植物材料的微生物的酶和其他蛋白质。本研究将研究森林土壤中的蛋白质分解，这是将有机氮转化为植物可用形式的主要瓶颈。蛋白质被细菌和真菌产生的酶（蛋白酶）分解并释放到土壤环境中。蛋白质与土壤矿物和有机组分之间的相互作用可以影响微生物蛋白酶的活性。这项研究将直接测量微生物和土壤对蛋白质降解的控制，从而更深入地了解植物氮素供应的瓶颈和氮素的其他环境命运。这项研究具有变革性的潜力，因为它将揭示耦合碳（C）和氮循环的核心机制。了解蛋白酶如何去磷和回收有机氮需要更准确地模拟有限的自然氮的可用性如何影响C-N循环之间的相互作用。研究将通过四项超出实验室范围的活动传达给科学界代表性不足的群体：（1）在安德鲁斯长期生态研究（LTER）网站举行的年度HJA日，该活动接触到大量公众;（2）协助每年夏季为初中和高中教师教授的土壤科学短期课程;（3）提供一名具有研究经验的K-12教师;（4）为研究生设计环境蛋白质组学的课程并进行短期教学。有机氮周转一直被认为是土壤生态系统氮素有效性的关键控制因素，但是将大分子N化合物分解成可同化的N-单体的方法却很少受到关注。拟议的研究将检查有机氮的周转，特别是蛋白质，这构成了土壤有机氮的最大部分。土壤氮循环的一个新的概念性的方法，提出了承认“矩阵”和“微生物”控制的有机氮的生物利用度：与土壤基质控制的蛋白质氮的可及性与一个复杂的微生物群落在响应环境刺激产生的蛋白酶的多样性互补的化学和物理过程的相互作用。该模型将通过两个目标进行探索：（1）确定两种15 N标记的“底物”蛋白质在土壤中的命运，这些蛋白质具有不同的理化特性，跨越资源可用性、矿物学和微生物生态学的梯度，特别强调蛋白质-矿物和蛋白质-有机质相互作用作为基质调节贡献者的相对重要性;（2）测定不同氮素有效性土壤中不同微生物类群对蛋白酶活性的相对贡献，并通过C与N限制对蛋白酶活性的控制，探索蛋白质周转的微生物调节。与这些目标相关的研究问题将使用温带森林中特征良好，长期实验的土壤来解决。15 N标记的蛋白质将用于确定其周转率以及蛋白质中C和N的命运。这将与蛋白酶的表征和其活性的测量相结合。将进行操作实验以确定细菌和真菌对蛋白酶活性的相对贡献。这些数据将提供深入了解控制机制在我们的概念模型中的有机氮周转。总的来说，目标1和2中产生的数据将使我们能够确定土壤基质和微生物群落对蛋白质N在土壤中降解时的命运的机械功能。它将揭示微生物蛋白酶的活性如何在土壤生态系统中变化，并通过其催化类型来表征它们。了解蛋白酶的性质和行为对于协调它们如何获得不同形式的土壤相关蛋白至关重要，因此构成了未来研究成功的基本前提。为此，拟议的研究将产生土壤中氮转化和保留的定量信息，这对土壤生产力和环境健康的管理也至关重要。拟议的研究所产生的数据和功能关系最终旨在耦合到N循环模型，分离微生物功能组或酶的活性。这项研究的成果将包括一个更定量的了解微生物蛋白酶在陆地生态系统中的有机氮循环的作用和蛋白酶的来源和多样性。

Plant productivity in natural and managed ecosystems is often limited by nitrogen (N), which plants obtain from the soil mainly as ammonium or nitrate - both of which are inorganic forms of N. Yet, more than 95% of the N in soil exists in organic forms, primarily enzymes and other proteins that originated from decaying plant material or from the microorganisms that decompose it. This research will study protein breakdown in forest soil, which is the major bottleneck in converting organic N into forms available to plants. Proteins are broken down by enzymes (proteases) that are produced and released into the soil environment by bacteria and fungi. Interactions between proteins and soil-mineral and organic components can affect the activity of microbial proteases. This research will directly measure microbial and soil controls on protein degradation and thus provide a deeper understanding of the bottleneck in the supply of N to plants and other environmental fates of N. This research has the potential to be transformative in that it will reveal a core mechanism coupling carbon (C) and N cycling. Understanding how proteases de-polymerize and recycle organic N is needed to more accurately model how limited natural nitrogen availability affects interactions between C & N cycles. Research will be conveyed to groups underrepresented in science through four activities that extend beyond the laboratory: (1) the annual HJA Day at the Andrews Long-Term Ecological Research (LTER) site, which reaches large numbers of the general public; (2) assist in a short-course in soil science that is taught each summer for middle and high school teachers; (3) provide a K-12 teacher with research experience; and (4) design curriculum and teach a short-course in environmental proteomics for graduate students.Organic N turnover has long been recognized as key controller of N availability in soil ecosystems, but the processes that break down macromolecular N compounds into assimilable N-monomers have received little attention. The proposed research will examine the turnover of organic N, particularly proteins, which make up the greatest fraction of soil organic N. A new conceptual approach towards the soil N cycle is proposed that recognizes "matrix" and "microbial" controls on the bioavailability of organic N: Interacting chemical and physical processes associated with the soil matrix control the accessibility of proteinaceous N to a diverse complement of proteases produced by a complex microbial community in response to environmental stimuli. This model will be explored through two objectives: (1) determining the fate of two 15N-labeled 'substrate' proteins with distinct physicochemical characteristics in soils across a gradient of resource availability, mineralogy, and microbial ecology, with special emphasis on the relative importance of protein-mineral and protein-organic matter interactions as contributors to matrix regulation; (2) determining the relative contributions of different microbial groups to protease activity in soils that vary in N availability, and the control of protease activity by C versus N limitation as a means of exploring the microbial regulation of protein turnover. Research questions associated with these objectives will be addressed using soils of well-characterized, long-standing experiments in temperate forests. 15N-labeled proteins will be used to determine their turnover rates and the fate of the C and N in the proteins. This will be coupled with characterization of proteases and measurements of their activities. A manipulative experiment will be done to determine the relative contribution to protease activity by bacteria and fungi. These data will provide insight into the control mechanisms functioning in our conceptual model of organic N turnover. Collectively, data generated in Objectives 1 and 2 will allow us to identify the mechanistic functions that the soil matrix and microbial community exert on the fate of protein N as it is degraded in soils. It will reveal how the activities of microbial proteases vary among soil ecosystems and characterize them by their catalytic types. Knowing the properties and behavior of proteases is essential to reconcile how they access the different forms of soil-associated proteins, and thus constitutes a fundamental prerequisite for successful future studies. To this end, the proposed research will generate quantitative information of N transformation and retention in soil, which is also critical to the management of soil productivity and environmental health. The data and functional relationships generated by the proposed research are eventually intended to be coupled to N cycling models that separate the activity of microbial functional groups or enzymes. The outcomes of this research will include a more quantitative understanding of the role of microbial proteases in organic N cycling in terrestrial ecosystems and of the sources and diversity of proteases.